DLX6-AS1 regulates odonto/osteogenic differentiation in dental pulp cells under the control of BMP9 via the miR-128-3p/MAPK14 axis: A laboratory investigation.

AIM: The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal-like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6-AS1) in odonto/osteogenic differentiation induced by BMP9. METHODOLOGY: Custom RT2 profiler PCR array, quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of DLX6-AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t-test or one-way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p-value of less than .05 considered statistically significant. RESULTS: DLX6-AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR-128-3p participated in BMP9-induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR-128-3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6-AS1. Lastly, miR-128-3p directly interacted with both MAPK14 and DLX6-AS1. CONCLUSIONS: DLX6-AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR-128-3p/MAPK14 axis.

Effect of 3D-printed polycaprolactone/osteolectin scaffolds on the odontogenic differentiation of human dental pulp cells.

Cell-based tissue engineering often requires the use of scaffolds to provide a three-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds. Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PCL scaffolds with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blot was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PCL scaffolds with pores was significantly higher than to PCL scaffolds without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PCL scaffolds, but there was no statistically significant difference. 3D-printed PCL scaffolds with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs.

Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation.

Tissue regeneration therapy based on human dental pulp cells (hDPCs) faces the distinct challenge of cellular senescence during massive expansion in vitro. To further explore the regulatory mechanism of cellular senescence in hDPCs, we conduct experiments on young cells (Passage 5, P5) and replicative senescent (Passage 12, P12) hDPCs. The results confirm that hDPCs undergo replicative senescence with passaging, during which their ability to proliferate and osteogenic differentiation decreases. Notably, during replicative senescence, phosphoglycerate dehydrogenase (PHGDH), the key enzyme of the serine synthesis pathway (SSP), is significantly downregulated, as well as S-adenosylmethionine (SAM) levels, resulting in reduced H3K36me3 modification on Sirtuin 1 (SIRT1)and Runt-related transcription factor 2 (RUNX2) promoters. Inhibition of PHGDH leads to the same phenotype as replicative senescence. Serine supplementation fails to rescue the senescence phenotype caused by replicative senescence and inhibitors, in which folate metabolism-related genes, including serine hydroxymethyl transferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 1(MTHFD1), methylenetetrahydrofolate dehydrogenase 2(MTHFD2), are notably decreased. Our research raised a possibility that PHGDH may be involved in cellular senescence by affecting folate metabolism and histone methylation in addition to serine biosynthesis, providing potential targets to prevent senescence.

Anti-inflammatory cerium-containing nano-scaled mesoporous bioactive glass for promoting regenerative capability of dental pulp cells.

AIMS: This study aimed to investigate the anti-inflammatory and odontoblastic effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on dental pulp cells as novel pulp-capping agents. METHODOLOGY: Ce-MBGNs were synthesized using a post-impregnation strategy based on the antioxidant properties of Ce ions and proposed the first use of Ce-MBGNs for pulp-capping application. The biocompatibility of Ce-MBGNs was analysed using the CCK-8 assay and apoptosis detection. Additionally, the reactive oxygen species (ROS) scavenging ability of Ce-MBGNs was measured using the 2,7-Dichlorofuorescin Diacetate (DCFH-DA) probe. The anti-inflammatory effect of Ce-MBGNs on THP-1 cells was further investigated using flow cytometry and quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the effect of Ce-MBGNs on the odontoblastic differentiation of the dental pulp cells (DPCs) was assessed by combined scratch assays, RT-qPCR, western blotting, immunocytochemistry, Alizarin Red S staining and tissue-nonspecific alkaline phosphatase staining. Analytically, the secretions of tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Ce-MBGNs were confirmed to effectively scavenge ROS in THP-1-derived macrophages and DPCs. Flow cytometry and RT-qPCR assays revealed that Ce-MBGNs significantly inhibited the M1 polarization of macrophages (Mφ). Furthermore, the protein levels of TNF-α and IL-1ß were downregulated in THP-1-derived macrophages after stimulation with Ce-MBGNs. With a step-forward virtue of promoting the odontoblastic differentiation of DPCs, we further confirmed that Ce-MBGNs could regulate the formation of a conductive immune microenvironment with respect to tissue repair in DPCs, which was mediated by macrophages. CONCLUSIONS: Ce-MBGNs protected cells from self-produced oxidative damage and exhibited excellent immunomodulatory and odontoblastic differentiation effects on DPCs. As a pulp-capping agent, this novel biomaterial can exert anti-inflammatory effects and promote restorative dentine regeneration in clinical treatment. We believe that this study will stimulate further correlative research on the development of advanced pulp-capping agents.

Mitochondrial reactive oxygen species promote mitochondrial damage in high glucose-induced dysfunction and apoptosis of human dental pulp cells.

Background/purpose: High glucose (HG)-induced aberrant proliferation, apoptosis and odontoblastic differentiation of dental pulp cells (DPCs) have been implicated in the pathogenesis of impaired diabetic pulp healing; however, the underlying mechanism remains unclear. This study aimed to investigate the role of mitochondrial reactive oxygen species (mtROS) and mitochondria in HG-induced dysfunction and apoptosis of DPCs. Materials and methods: Human DPCs (hDPCs) were cultured in a low-glucose, high-glucose, mannitol, and MitoTEMPO medium in vitro. Methylthiazol tetrazolium assay, Annexin V-FITC/PI staining and scratch-wound assay were used to analyze cell proliferation, apoptosis and migration, respectively. Alkaline phosphatase staining and alizarin red S staining were used to evaluate cell differentiation. DCF-DA staining, MitoSOX staining, MitoTracker Red staining, JC-1 staining, and adenosine triphosphate (ATP) kit assay were performed to investigate total ROS and mtROS generation, mitochondrial density, mitochondrial membrane potential (MMP), and ATP synthesis, respectively. Quantitative PCR assay was performed to detect the mRNA expression of mitochondrial biogenesis- and dynamics-related markers. Transmission electron microscopy was used to observe the mitochondrial ultrastructure. Results: HG augmented the production of total ROS and mtROS, and triggered mitochondrial damage in hDPCs, as reflected by decreased mitochondrial density, depolarized MMP, reduced ATP synthesis, altered mRNA expression of mitochondrial biogenesis- and dynamics-related markers, and abnormal mitochondrial ultrastructure. Supplementation of MitoTEMPO alleviated the mitochondrial damage and reversed the aberrant proliferation, apoptosis, migration and odontoblastic differentiation of HG-stimulated hDPCs. Conclusion: HG triggers mitochondrial damage via augmenting mtROS generation, resulting in the inhibited proliferation, migration, and odontoblastic differentiation of hDPCs and enhanced their apoptosis.

Melatonin protects TEGDMA-induced preodontoblast mitochondrial apoptosis via the JNK/MAPK signaling pathway.

Resin monomer-induced dental pulp injury presents a pathology related to mitochondrial dysfunction. Melatonin has been regarded as a strong mitochondrial protective bioactive compound from the pineal gland. However, it remains unknown whether melatonin can prevent dental pulp from resin monomer-induced injury. The aim of this study is to investigate the effects of melatonin on apoptosis of mouse preodontoblast cells (mDPC6T) induced by triethylene glycol dimethacrylate (TEGDMA), a major component in dental resin, and to determine whether the JNK/MAPK signaling pathway mediates the protective effect of melatonin. A well-established TEGDMA-induced mDPC6T apoptosis model is adopted to investigate the preventive function of melatonin by detecting cell viability, apoptosis rate, expressions of apoptosis-related proteins, mitochondrial ROS (mtROS) production, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) level. Inhibitors of MAPKs are used to explore which pathway is involved in TEGDMA-induced apoptosis. Finally, the role of the JNK/MAPK pathway is verified using JNK agonists and antagonists. Our results show that melatonin attenuates TEGDMA-induced mDPC6T apoptosis by reducing mtROS production and rescuing MMP and ATP levels. Furthermore, mitochondrial dysfunction and apoptosis are alleviated only by the JNK/MAPK inhibitor SP600125 but not by other MAPK inhibitors. Additionally, melatonin downregulates the expression of phosphorylated JNK and counteractes the activating effects of anisomycin on the JNK/MAPK pathway, mimicking the effects of SP600125. Our findings demonstrate that melatonin protects mDPC6T cells against TEGDMA-induced apoptosis partly through JNK/MAPK and the maintenance of mitochondrial function, offering a novel therapeutic strategy for the prevention of resin monomer-induced dental pulp injury.

Notch signaling regulates mineralization via microRNA modulation in dental pulp stem cells.

OBJECTIVES: This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs. METHODS: DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression. RESULTS: Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-ß, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs. CONCLUSIONS: Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials.

A leptin-loaded poly-ϵ-caprolactone 3D printing scaffold for odontoblastic differentiation in human dental pulp cells.

This study investigated the effects on odontoblast differentiation of a 3D-printed poly-ϵ-caprolactone (PCL) scaffold that incorporated leptin. Material extrusion-type 3D printing with a 43 000-molecular weight PCL material was used to fabricate a PCL scaffold with a 6 mm diameter, 1 mm height, and 270-340 µm pore size. The experimental groups were PCL scaffolds (control group), PCL scaffolds with aminated surfaces (group A), and PCL scaffolds with leptin on the aminated surface (group L). The aminated surface was treated with 1,6-hexanediamine and verified by ninhydrin analysis. Leptin loading was performed using Traut's reagent and 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC). Groups A and L showed significantly higher surface wettability, pulp cell adhesion, and proliferation than the control group. Group L exhibited increased alkaline phosphatase, calcification deposits, and mRNA and protein expression of dentin sialophosphoprotein and dentin matrix acidic phosphoprotein 1 compared with the control group. In this study, a 3D-printed PCL scaffold containing leptin was enhanced odontoblast differentiation and dental pulp cells adhesion and proliferation.

LncRNA CARMN facilitates odontogenic differentiation of dental pulp cells by impairing EZH2.

OBJECTIVE: This study aimed to reveal the potential role of CARMN in odontogenic differentiation of dental pulp cells (DPCs). METHODS: Laser capture microdissection was used to detect Carmn in DPCs and odontoblasts in P0 mice. After manipulating CARMN expression in odontogenic differentiation induced hDPCs, the state of odontogenic differentiation was evaluated by ALP staining, ARS, and related marker expression in qRT-PCR and western blotting. The subcutaneous transplantation of HA/ß-TCP loaded with hDPCs was performed to verify CARMN's role in promoting odontogenic differentiation in vivo. RNAplex and RIP were employed to reveal potential mechanism of CARMN in hDPCs. RESULTS: CARMN expressed more abundantly in odontoblasts than DPCs in P0 mice. CARMN expression boosted during in vitro odontogenic differentiation of hDPCs. CARMN overexpression enhanced odontogenic differentiation of hDPCs in vitro, while inhibition impaired the process. CARMN overexpression in HA/ß-TCP composites promoted more mineralized nodule formation in vivo. CARMN knockdown led to soared EZH2, while CARMN overexpression brought about EZH2 inhibition. CARMN functioned via direct interaction with EZH2. CONCLUSIONS: The results uncovered CARMN as a modulator during the odontogenic differentiation of DPCs. CARMN promoted odontogenic differentiation of DPCs by impairing EZH2.

Human Digested Dentin Matrix for Dentin Regeneration and the Applicative Potential in Vital Pulp Therapy.

INTRODUCTION: Human dentin is a natural acellular matrix with excellent reported biocompatibility. The aim was to fabricate a novel dentin matrix material from human dentin and investigate its applicative potential for vital pulp therapy. METHODS: Digested dentin matrix extract (DDME) was fabricated using controlled enzymatic digestion under acidic conditions. The surfaces and biocompatibility of DDME were then investigated, with its effects on the odontogenic differentiation of human dental pulp cells (hDPCs) also studied. The ability of DDME to induce mineralization was assessed in a nude mouse model. The performance of DDME as a pulp capping agent was evaluated in an in situ rat model. The molecular mechanism was verified by mRNA sequencing. RESULTS: A novel type of dentin matrix material with a uniform size of 8 µm was fabricated. DDME had a similar band compared with grinded dentin matrix, with a smaller size, and more uneven surface, as detected by Fourier-transform infrared spectrometer and X-ray photoelectron spectroscopy. DDME at low concentrations did not affect hDPC viability or proliferation, but enhanced runt-related transcription factor 2, dentin matrix acidic phosphoprotein 1, and COL1A1 (collagen type I alpha 1 chain) expression in hDPCs in vitro. DDME was superior to HA-TCP (hydroxyapatite-tricalcium phosphate) in dentin-like mineralized tissue formation after subcutaneous transplantation. In the rat model of pulpotomy, DDME showed visible curative effects. The underlying mechanism may be the inhibition of Hippo signaling following DDME treatment. DDME promoted Yes-associated protein (YAP) 1 nuclear influx, thereby enhancing the expression of DMP-1 (dentin matrix acidic phosphoprotein 1), which was reversed by YAP inhibitor treatment. CONCLUSIONS: Human DDME can be used as a biomaterial for dentin regeneration. The combined application of DDME and current pulp capping agents is a potential choice for vital pulp therapy.

MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

Tideglusib enhances odontogenic differentiation in human dental pulp stem cells in vitro.

AIM: Tideglusib is a small molecule agonist of the canonical Wnt pathway. The present study investigated the influence of Tideglusib on human dental pulp stem cell (hDPSC) proliferation, apoptosis, migration and odonto/osteogenic differentiation. METHODOLOGY: hDPSCs were treated with 50, 100 nM or 200 nM Tideglusib. ß-catenin accumulation was detected by immunofluorescence staining. Colony-forming unit ability was assessed by staining with Coomassie blue. Cell cycle progression and cell apoptosis were investigated using flow cytometry. Cell migration was examined using an in vitro wound-healing assay. Osteogenic differentiation was examined using alkaline phosphatase (ALP) staining, alizarin red S staining and osteogenic-related gene expression. The gene expression profile was examined using a high-throughput RNA sequencing technique. All experiments were repeated using cells derived from at least four different donors (n = 4). The Mann-Whitney U-test was used to identify significant differences between two independent group comparisons. For three or more group comparisons, statistical differences were assessed using the Kruskal-Wallis test followed by pairwise comparison. The significance level was set at 5% (p < .05). RESULTS: Tideglusib activated the Wnt signalling pathway in hDPSCs as demonstrated by an increase in cytoplasmic ß-catenin accumulation and nuclear translocation. Tideglusib did not affect hDPSC proliferation, cell cycle progression, cell apoptosis or cell migration. In contrast, 50 and 100 nM Tideglusib significantly enhanced mineralization and osteogenic marker gene expression (RUNX2, ALP, BMP2 and DSPP; p < .05). CONCLUSIONS: Tideglusib enhanced the odonto/osteogenic differentiation of hDPSCs. Therefore, incorporating this bioactive molecule in a pulp-capping material could be a promising strategy to promote dentine repair.

Dose-dependent effects of neem crude extract on human dental pulp cell and murine osteoblast viability and mineralization

Abstract Neem has been used as a medicine due to its beneficial properties such as anti-microbial effects. Neem products for oral application are on the rise. Before recommendation for therapeutic use in human, its effects on cellular activities need to be examined. Therefore, the aim of this study was to test the effects of the ethanolic neem crude extract on dental pulp cells and osteoblasts in terms of cell viability, mineralization, and gene expressions. The ethanolic neem extract derived from dry neem leaves was subjected to chemical identification using GC-MS. Human dental pulp stem cells (hDPSCs) and pre-osteoblasts (MC3T3) were treated with various concentrations of the neem crude extract. Cell viability, mineralization, and gene expressions were investigated by MTT assay, real-time PCR, and alizarin red S assay, respectively. Statistical analysis was performed by one-way ANOVA followed by Dunnett test. GC-MS detected several substance groups such as sesquiterpene. Low to moderate doses of the neem crude extract (4 - 16 µg/ml) did not affect hDPSC and MC3T3 viability, while 62.5 µg/ml of the neem extract decreased MC3T3 viability. High doses of the neem crude extract (250 - 1,000 µg/ml) significantly reduced viability of both cells. The neem crude extract at 1,000 µg/ml also decreased viability of differentiated hDPSC and MC3T3 and their mineralization. Furthermore, 4 µg/ml of neem inhibited viability of differentiated hDPSC. There is no statistical difference in gene expressions related to cell differentiation. In conclusion, the neem crude extract affected cell viability and mineralization. Cell viability altered differently depending on the doses, cell types, and cell stages. The neem crude extract did not affect cell differentiation. Screening of its effect in various aspects should be examined before the application for human use.

Resumo O Neem tem sido utilizado como medicamento devido às suas propriedades benéficas, tais como os efeitos antimicrobianos. Os produtos Neem para aplicação oral estão a aumentar. Antes da recomendação para uso terapêutico no ser humano, os seus efeitos nas atividades celulares precisam ser examinados. Por conseguinte, o objectivo deste estudo era testar os efeitos do extracto bruto de neem etanólico nas células de polpa dentária e osteoblastos em termos de viabilidade celular, mineralização e expressões genéticas. O extracto de neem etanólico derivado de folhas secas de neem foi sujeito a identificação química utilizando GC-MS. As células estaminais de polpa dentária humana (hDPSCs) e os pré-osteoblastos (MC3T3) foram tratados com várias concentrações do extrato bruto de neem. A viabilidade celular, mineralização, e expressões genéticas foram investigadas pelo ensaio MTT, PCR em tempo real, e o ensaio S vermelho de alizarina, respectivamente. A análise estatística foi realizada por ANOVA unidirecional seguida pelo teste Dunnett. O GC-MS detectou vários grupos de substâncias como o esquisterpeno. Doses baixas a moderadas do extracto bruto de neem (4 - 16 µg/ml) não afetaram a viabilidade do hDPSC e MC3T3, enquanto 62,5 µg/ml do extracto de neem diminuiu a viabilidade do MC3T3. Doses elevadas do extrato bruto de neem (250 - 1.000 µg/ml) reduziram significativamente a viabilidade de ambas as células. O extrato bruto de neem a 1.000 µg/ml também diminuiu a viabilidade de hDPSC e MC3T3 diferenciados e a sua mineralização. Além disso, 4 µg/ml de neem inibiu a viabilidade do hDPSC diferenciado. Não há diferença estatística nas expressões genéticas relacionadas com a diferenciação celular. Em conclusão, o extrato bruto do neem afetou a viabilidade celular e a mineralização. A viabilidade celular alterou-se diferentemente dependendo das doses, tipos de células, e fases celulares. O extrato bruto do neem não afetou a diferenciação celular. O rastreio do seu efeito em vários aspectos deve ser examinado antes da aplicação para uso humano.

Odontogenic Effect of Icariin on the Human Dental Pulp Cells.

Background and Objectives: Human dental pulp cells (HDPCs) can be used for dentin regeneration due to its odontogenic differentiation property. Icariin can induce osteogenic differentiation of stem cells. However, its potential to induce odontogenic differentiation of HDPCs remains unclear. Thus, the aim of this study was to evaluate the capacity of icariin to induce odontogenic differentiation of HDPCs and investigate the underlying molecular mechanism. Materials and Methods: Cell viability assay was used to detect the cytotoxicity of icariin to HDPCs. Effect of icariin on HDPCs chemotaxis was measured by scratch migration assay. The mineralized and odontogenic differentiation of HDPCs was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, real-time PCR, and Western blot of dentin matrix protein 1 (DMP 1) and dentin sialophosphoprotein (DSPP). In addition, Mitogen-activated protein kinase (MAPK) signaling pathway of icariin-induced biomineralization was investigated by Western blot. Results: Cells treated with icariin at all concentrations tested maintained viability, indicating that icariin was biocompatible. Icariin accelerated HDPCs chemotaxis (p < 0.05). Expression levels of related odontogenic markers were increased in the presence of icariin (p < 0.05). Icariin-induced odontogenic differentiation occurred via activation of the MAPK signaling pathway. Furthermore, MAPK inhibitors suppressed expression levels of DSPP and DMP 1 protein, ALP activity, and mineralization of HDPCs. Conclusions: Icariin can upregulate odontogenic differentiation of HDPCs by triggering the MAPK signaling pathway.

Nell-1 attenuates lipopolysaccharide-induced inflammation in human dental pulp cells.

Nel-like molecule type 1 (Nell-1) is a secreted protein that plays an important role in osteoinduction in multiple animal models. A previous study has suggested the anti-inflammatory effect of Nell-1 on bone inflammation inhibition. However, its role in pulpitis has not been investigated. The present study aims to explore the effect of human recombinant Nell-1 (Nell-1) on rat pulp inflammation response, and its effect on lipopolysaccharide-induced inflammation in human dental pulp cells and its related intracellular signaling pathways. 30 Wistar rats with healthy non-carious maxillary first molars were chosen, Nell-1 was absorbed onto a sterile collagen sponge and capped onto exposed pulps. The expression of IL-6 and IL-8 were detected by immunohistochemical staining. Human dental pulp cells (hDPCs) were isolated from healthy extracted premolars and third molars. hDPCs were co-cultured with Escherichia coli lipopolysaccharide (LPS), Nell-1 protein, and mitogen-activated protein kinase (MAPK) inhibitors. The expression of pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8, was examined via quantitative real-time PCR and enzyme-linked immunosorbent assay. The results showed that Nell-1 inhibited the inflammatory response of rat pulp. LPS treatment contributed to the expression of inflammatory factors in hDPCs, whereas Nell-1 obviously suppressed the LPS-induced inflammation. p38 MAPK and extracellular signal-regulated kinase (ERK) MAPK inhibitors attenuated the anti-inflammatory effect of hrNell-1, whereas the c-Jun N-terminal kinases (JNK) MAPK inhibitor exerted minimal effect. Therefore Nell-1 could inhibit LPS-induced inflammation in human dental pulp cells, and this effect may be mediated by p38 and ERK MAPK signaling pathways, but not JNK MAPK signaling pathway.

Investigation of in vitro odonto/osteogenic capacity of cannabidiol on human dental pulp cell.

INTRODUCTION: Vital pulp treatment (VPT) maintains tooth vitality with certain dental materials by protecting pulp from noxious stimulation and promoting repair through enhancing cell proliferation/differentiation, migration, and inducing odontogenesis. As a non-psychotropic cannabis constituent, cannabidiol (CBD) possesses the properties of analgesic, anti-inflammation, and osteogenesis. Therefore, we hypothesize that CBD may induce the odonto/osteogenesis of human dental pulp cells (HDPCs), a critical feature using as effective pulp capping agent for VPT. MATERIALS AND METHODS: In this in vitro study, the cytotoxicity of CBD on HDPCs was determined by MTT assay. Scratch assay was performed to analyze HDPC migration. The biomineralization was examined by collagen synthesis and calcium nodule formation and related odonto/osteogenic and angiogenic genes. Cannabinoid receptor (CB) specificity was evaluated by Western blotting and Von Kossa staining using specific antagonists AM251 for cannabinoid receptor 1 (CB1) and AM 630 targeted at cannabinoid receptor 2 (CB2). In addition, the underlying molecular mechanism of CBD-induced biomineralization were investigated by examining CB-dependent MAPK signaling pathways. RESULTS: CBD demonstrated bi-phasic effects on HDPC viability in tested concentrations. We found CBD significantly promoted cell migration, enhanced collagen synthesis and mineralized deposits in HDPCs when treated by 1 µM CBD supplemented in the differentiation media. RT-PCR revealed CBD increased the expression of angiogenic and odontogenic genes, such as DSPP, DMP-1, OPN, ALP, Runx2, VEGFR1 and ICAM-1. These effects were via MAPK activation in a manner mainly mediated by CB2. CONCLUSION: The results from this study suggested that CBD can induce odonto/osteogenesis from HDPCs and has the potential to develop new therapeutics in VPT in dentistry.

JNK-mediated blockage of autophagic flux exacerbates the triethylene glycol dimethacrylate-induced mitochondrial oxidative damage and apoptosis in preodontoblast.

Mitochondrial dependent oxidative stress (OS) and subsequent cell death are considered as the major cytotoxicity caused by Triethylene glycol dimethacrylate (TEGDMA), a commonly monomer of many resin-based dental composites. Under OS microenvironment, autophagy serves as a cell homeostatic mechanism and maintains redox balance through degradation or turnover of cellular components in order to promote cell survival. However, whether autophagy is involved in the mitochondrial oxidative damage and apoptosis induced by TEGDMA, and the cellular signaling pathways underlying this process remain unclear. In the present study, we demonstrated that TEGDMA induced mouse preodontoblast cell line (mDPC6T) dysfunctional mitochondrial oxidative response. In further exploring the underlying mechanisms, we found that TEGDMA impaired autophagic flux, as evidenced by increased LC3-II expression and hindered p62 degradation, thereby causing both mitochondrial oxidative damage and cell apoptosis. These results were further verified by treatment with chloroquine (autophagy inhibitor) and rapamycin (autophagy promotor). More importantly, we found that the JNK/MAPK pathway was the key upstream regulator of above injury process. Collectively, our finding firstly demonstrated that TEGDMA induced JNK-dependent autophagy, thereby promoting mitochondrial dysfunction-associated oxidative damage and apoptosis in preodontoblast.

LncRNA MALAT1 Functions as a Competing Endogenous RNA to Regulate BMI1 Expression by Sponging miR-200c/miR-203 in the Control of the Differentiation of Pulp Cells.

BACKGROUND: Long non-coding RNAs (lncRNAs) and miRNAs (microRNAs) are considered as key regulators of several biological processes, including dental development. In this study, we explored the lncRNAs and miRNAs which are involved in dental development. METHOD: Real-time PCR was performed to identify the candidate lncRNAs and miRNAs involved in dental development. Bioinformatics analysis and luciferase assay were carried out to establish the regulatory relationships between MALAT1, miR-203 and miR-200c in dental development. RESULTS: Among all candidate lncRNAs, only MALAT1 was highly expressed in differentiated human dental pulp cells (hDPCs), and among all candidate miRNAs which are down-regulated in differentiated hDPCs, miR-203, and miR-200c are most decreased. Furthermore, MALAT1 was up-regulated while miR-203 and miR-200c were down-regulated in differentiated hDPCs in a time-dependent manner. MiR-203 and miR-200c were proved to bind to MALAT1. Moreover, BMI1 was identified as a target gene of miR-203 or miR-200c, and BMI1 was time-dependently decreased in hDPCs cultured with odontogenic medium. On the contrary, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), osteocalcin (OCN), and alkaline phosphatase (ALP), were time-dependently increased in hDPCs cultured with odontogenic medium. Finally, the overexpression of MALAT1 and the knockdown of miR-203/miR-200c both significantly increased the levels of BMI1, DSPP, DMP-1, OCN, and ALP, while the effect of knockdown of miR-203/miR-200c was much stronger than that of the overexpression of MALAT1. CONCLUSION: Our results demonstrated that MALAT1 functions as a competing endogenous RNA of miR-203 and miR-200c and accordingly promotes BMI1 expression. Therefore, MALAT1 may serve as a biomarker for dental development.

Evaluation of Cannabinoids on the Odonto/Osteogenesis in Human Dental Pulp Cells In Vitro.

INTRODUCTION: Cannabinoids possess anti-inflammatory, analgesic, and osteogenic effects in different cell types and tissues. The null hypothesis is delta-9-tetrahydrocannabinol (THC) might induce dental tissue repair and regeneration. The aim of this study was to investigate the effect of THC on human dental pulp cell (HDPC) viability and biomineralization as well as the molecular mechanism of THC-induced odonto/osteogenic differentiation of HDPCs. METHODS: The toxicity of THC on HDPCs was determined by 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. The odonto/osteogenic differentiation marker genes of HDPCs were assessed by real-time polymerase chain reaction with or without THC treatment. HDPC biomineralization was examined by collagen synthesis and calcium nodule deposition. The molecular mechanism of THC on HDPCs was investigated by examining the mitogen-activated protein kinase (MAPK) signaling pathway via blocking cannabinoid receptor type 1 or 2 receptors. RESULTS: We found that THC had no inhibition of HDPC vitality in the testing concentration (0-100 µmol/L). THC showed biphasic effects on HDPC proliferation. At a low dose (<5 µmol/L), THC considerably increased HDPC cell division. HDPC proliferation reduced with higher THC concentrations (>5 µmol/L). The expression of odonto/osteogenic marker genes were up-regulated in the presence of cannabinoids. These were confirmed by increased collagen synthesis and mineralized calcium nodule formation in the cannabinoid group. The effect of THC-induced odonto/osteogenesis occurred via MAPK signaling. CONCLUSIONS: THC was biocompatible to HDPCs by promoting their mitogenic division in a biphasic pattern depending on the concentration. THC induced HDPC odonto/osteogenic differentiation through the activation of MAPK mediated by CB1 and CB2 receptors. Cannabinoids may play an important role in the HDPC regeneration process and potentially be used as a pulp-capping agent.

Inhibition of carrageenan-induced dental inflammatory responses owing to decreased TRPV1 activity by Dexmedetomidine.

BACKGROUND: Dexmedetomidine (Dex) is a highly selective agonist of the α2 adrenergic receptor and a common sedative; however, its anti-inflammatory effect has been studied. In this study, the inhibitory effect of Dex on inflammation in dental pulp cells was assessed. For this, the effect of Dex on inflammation induced by carrageenan (Car) in human dental pulp cells (hDPCs) was evaluated. Car incubation induced a robust inflammatory response in hDPCs as well as activation of PKA-STAT3 and PKC-nuclear factor kappa B (NF-κB) signaling pathways. RESULTS: Dex reduced the expression of inflammatory cytokines in a dose-dependent manner. Meanwhile, the phosphorylation of PKA, PKC, STAT3, and NF-κB as well as the nuclear accumulation of STAT3 and NF-κB were significantly increased in Dex-treated Car-induced hDPCs. Western blotting results also showed that the phosphorylation level of transient receptor potential cation channel subfamily V member 1 (TRPV1) was downregulated as a result of Dex treatment. Furthermore, we found that administration of the TRPV1 agonist capsaicin (Cap) reversed the effects of Dex on proinflammatory cytokines; however, the expression and activation of PKA-STAT3 and PKC-NF-κB signals were not altered owing to Cap administration. CONCLUSIONS: These results indicate that Dex plays a defensive role in dental pulp inflammation by regulating the TRPV1 channel and can be used as a potential target for human dental pulp inflammation intervention.